CN116042422B - Yeast-derived exosome and preparation method and application thereof - Google Patents
Yeast-derived exosome and preparation method and application thereof Download PDFInfo
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- CN116042422B CN116042422B CN202310096587.XA CN202310096587A CN116042422B CN 116042422 B CN116042422 B CN 116042422B CN 202310096587 A CN202310096587 A CN 202310096587A CN 116042422 B CN116042422 B CN 116042422B
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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Abstract
The invention provides a yeast-derived exosome, and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) When yeast is cultured to a logarithmic phase, performing simulated environment sub-stimulus treatment, adding amino acid, and then continuously culturing to a stationary phase to obtain fermentation liquor; (2) Carrying out solid-liquid separation on the fermentation liquor to obtain an extracellular body; mixing the extracellular exosomes with saccharides to obtain the yeast-derived exosomes. The invention stimulates the yeast cells by adopting the sub-stimulus of the simulation environment, the yeast cells generate stress response, and the secreted high-activity exosomes have good anti-inflammatory and antioxidant effects.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a yeast-derived exosome, and a preparation method and application thereof.
Background
Delaying skin aging is a permanent research subject, and the capacity of the anti-aging market in China continues to increase at a high speed in recent years. According to the investigation data of the Fubooth and the Eurononitor, the scale of the anti-aging market of the China in 2018 reaches 472 hundred million yuan, and the equivalent speed increase is up to 17.0%; the 2019 scale reaches 575 hundred million yuan, and the same ratio is increased by 21.8 percent, which accounts for 12 percent of the whole cosmetic market scale; and from 2016, the continuous acceleration trend is presented, and the compound acceleration reaches 11.8% in nearly five years, so that the method has a very good prospect. According to the data issued by the Chinese essence, perfume and cosmetic industry association, the most concerned and popular maintenance service project of females is anti-aging maintenance at present, the mention rate of the maintenance service project in all projects is up to 72.2%, and 90% of Chinese females take anti-aging measures. It was predicted that in 2021, the global anti-aging market size would reach 2160 billion dollars. The active ingredients of the common aging-delaying cosmetics in the market at present mainly comprise polypeptide, plant active polyphenol and the like, and the aging-delaying products mainly face the problems of single product combination, repeated market positioning, serious homogenization and the like.
Researchers find that yeast has strong stress response capability, and when the yeast is subjected to stress, such as ultraviolet irradiation, thermal stimulation or chemical injury, the yeast can induce and evolve a complete induction mechanism in the adaptation process, and a plurality of protective substances with self-repairing property can be automatically and rapidly synthesized to resist the injury effect caused by the oxidative injury.
Exosomes are biomolecular nanostructures released from cells, which carry specific biomolecular information, and since more than 30 years ago have been discovered, since their unique biological properties and functions have been studied without heat loss, recent studies have shown that exosomes of stem cells play a very important role in the anti-aging process of the body. Currently, there are hundreds of core factors known to stem cell exosomes to have anti-aging and skin-beautifying effects, such as cytokines FGF, EGF, cell signaling proteins Wnt4, wnt11, anti-aging mirnas: miRNA-124a, miRNA125b, miRNA21, miRNA-146a, miRNA-181c, miRNA-let-7b, etc. But at the same time, the industrialization and application of stem cells are limited. Yeast is a single-cell eukaryotic organism with protein content of 40% -60%, and protein hydrolysate thereof comprises eight amino acids necessary for human body, so that the preparation method and the efficacy of yeast exosomes are researched, and the yeast has great significance for promoting the application of yeast in the fields of medicines, foods, cosmetics and the like.
CN110777083a discloses an exosome extracted from yeast cells, which contains specific peptide fragment proteins inside. There is provided a method for extracting exosomes from yeast cells, comprising the steps of: culturing yeast cells, centrifuging after obtaining a cell culture, collecting supernatant, filtering, mixing the supernatant with a separating liquid, and centrifuging again to obtain a precipitate, namely yeast cell exosomes. The exosomes of the method are extracted from yeast cells, contain specific peptide protein, can be used as a marker of yeast based on the characteristics, and have great market prospect and application value in clinic.
CN114009754a discloses a preparation method of fermented plant exosomes, and the novel fermented plant exosomes of food raw materials are prepared through processes of enzymolysis, mixing, enzyme deactivation extraction, fermentation culture, centrifugation and the like. Different from the traditional preparation method of the plant exosomes, the fermentation of the plant exosomes is mainly realized through two important links of enzymolysis and fermentation, the optimal enzymolysis effect is exerted through the enzymolysis of an enzyme preparation at a specific temperature and time, and the fermentation of the fermentation fungus powder at the specific temperature and time reaches a certain acidity, so that the fermentation plant exosomes are obtained.
The prior art discloses a series of preparation methods of exosomes, but the exosomes cannot be stored for a long time due to high biological activity, so that development of exosomes which have high biological activity, can be stored for a long time and have good skin care efficacy is an important research point in the field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a yeast-derived exosome, a preparation method and application thereof, which can keep high bioactivity for a long time and can be used for relieving skin inflammation and preventing oxidation.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for preparing yeast-derived exosomes, the method comprising the steps of:
(1) When yeast is cultured to a logarithmic phase, performing simulated environment sub-stimulus treatment, adding amino acid, and then continuously culturing to a stationary phase to obtain fermentation liquor;
(2) Carrying out solid-liquid separation on the fermentation liquor to obtain an extracellular body; mixing the extracellular exosomes with saccharides to obtain the yeast-derived exosomes.
The invention adopts the simulation environment sub-stimulus to stimulate the yeast in the logarithmic phase, the yeast cells generate stress reaction, secrete high-activity exosomes, and simultaneously amino acid is added, so that the yeast can be compounded with other substances to play an antioxidant role more effectively; solid-liquid separation is carried out on the fermentation liquor, so that solid impurities are effectively removed, and exosomes with higher purity can be obtained; the extracted exosomes are mixed with the saccharides, so that the exosomes are high in biological activity and difficult to store, and after the exosomes are mixed with the saccharides, the storage time can be effectively prolonged while the biological activity is not reduced.
Preferably, the yeast is inoculated in an amount of 6 to 10%, for example, 7%, 8% or 9%.
Preferably, the yeast comprises any one or a combination of at least two of brewer's yeast, pichia pastoris or candida.
Preferably, the yeast is a combination of lager brewing yeast, pichia pastoris and candida.
Preferably, the candida is garcinia cambogia (GARCINIA CAMBOGIA).
Preferably, the inoculation ratio of the beer yeast, the Pichia pastoris and the candida is 1 (0.8-1.2): (0.8-1.2), and for example, the inoculation ratio can be 1:0.8:0.9, 1:0.9:0.9, 1:0.8:1, 1:1:0.9, 1:1:1:1.1:1.2, 1:1.2:1.1, and the like.
Preferably, the time from the cultivation to the logarithmic phase is 8 to 15 hours, and for example, 9 hours, 10 hours, 12 hours, 13 hours or 14 hours, etc. can be used.
Preferably, the time for continuing the culture to the stationary phase is 30-45 hours, and may be, for example, 32 hours, 35 hours, 38 hours, 42 hours, or the like.
Preferably, the temperature at which the cultivation is continued to the logarithmic phase and the stationary phase in step (1) is each independently 25 to 30℃and may be 26℃or 27℃or 28℃or 29℃or the like.
Other specific point values in the above numerical ranges are selectable, and will not be described in detail here.
Preferably, the simulated environmental sub-stimulus comprises any one of oxidative stress stimulus, high osmotic pressure stimulus, high temperature stimulus or low temperature stimulus.
Preferably, the reagent used for the oxidative stress stimulation is hydrogen peroxide.
Preferably, H in the medium upon stimulation of the oxidative stress 2 O 2 The content of (C) is 0.08-0.25mmol/L, for example, 0.1mmol/L, 0.13mmol/L, 0.16mmol/L, 0.2mmol/L, 0.24mmol/L, etc., preferably 0.2mmol/L.
Preferably, the agent used for the high osmotic pressure stimulation is a saline solution.
Preferably, the brine solution comprises an aqueous potassium chloride solution and/or an aqueous sodium chloride solution.
Preferably, the concentration of the salt in the medium at the time of the high osmotic pressure stimulation is 0.3 to 0.8mmol/L, for example, 0.4mmol/L, 0.5mmol/L, 0.6mmol/L, or 0.7mmol/L, etc., preferably 0.5mmol/L.
Preferably, the temperature of the high temperature stimulus is 37-40 ℃, for example, 38 ℃, 39 ℃ or 39.5 ℃ and the like.
Preferably, the temperature of the low temperature stimulus is 0 to 15 ℃, for example, 1 ℃,4 ℃, 8 ℃, 12 ℃ or the like.
Preferably, the amino acid comprises one or a combination of at least two of cysteine, methionine or cystine, preferably cysteine.
Preferably, the amino acid is added in an amount of 0.1 to 0.25% by mass of the medium, for example, 0.15%, 0.2% or 0.24% by mass, etc.
Preferably, the solid-liquid separation comprises the steps of centrifugation, primary filtration, concentration and secondary filtration.
Preferably, the centrifugation time is 8-12min, for example, 9min, 10min or 11 min; the rotation speed is 3000-6000rpm, for example 4000rpm, 4500rpm or 5000 rpm.
Preferably, the pore size of the filter membrane for one filtration is 80-100kDa, for example, 85kDa, 90kDa or 95kDa, etc.
Preferably, the pore size of the filter membrane for secondary filtration is 20-30kDa, for example, 23kDa, 26kDa or 29kDa, etc.
Preferably, the concentration is reduced to 1/6-1/4 of the volume, for example, 1/6, 1/5 or 1/4, etc.
Other specific point values in the above numerical ranges are selectable, and will not be described in detail here.
The solid-liquid separation is carried out by adopting centrifugation and twice filtration, the purpose of centrifugation is to remove culture medium impurities and cell fragments in fermentation liquor, supernatant is collected after the first filtration to effectively remove small molecules, the second filtration is carried out after concentration, and a filter membrane with smaller pore diameter is adopted to carry out filtration to effectively separate out solution, and the precipitate is reserved as an extracellular body.
Preferably, the saccharide is a algal polysaccharide solution.
Preferably, the seaweed polysaccharide solution contains 1-4% by mass of seaweed polysaccharide, for example, 2%, 2.5% or 3%.
Preferably, the mass ratio of the seaweed polysaccharide solution to the extracellular body is 1 (0.8-1.2), for example, 1:0.9, 1:1 or 1:1.1, etc.
Preferably, the mixing further comprises a step of freeze drying.
Preferably, the preparation method comprises the following steps:
(1) Culturing yeast at 25-30deg.C for 8-15 hr, performing simulation environment sub-stimulus treatment until logarithmic phase, adding amino acid, and culturing for 30-45 hr until stable phase to obtain fermentation broth;
the inoculation amount of the yeast is 6-10%, the yeast is a combination of beer yeast, pichia pastoris and candida, and the inoculation ratio of the beer yeast, the pichia pastoris and the candida is 1 (0.8-1.2): 0.8-1.2);
the simulation environment sub-stimulus comprises any one of oxidative stress stimulus, high osmotic pressure stimulus, high temperature stimulus or low temperature stimulus, wherein the reagent adopted by the oxidative stress stimulus is hydrogen peroxide, and H in the culture medium is used during the oxidative stress stimulus 2 O 2 The content of (2) is 0.08-0.25mmol/L; the high osmotic pressure stimulation adopts a saline solution, wherein the saline solution comprises a potassium chloride aqueous solution and/or a sodium chloride aqueous solution, and the concentration of salt in a culture medium is 0.3-0.8mmol/L during the high osmotic pressure stimulation; the temperature of the high-temperature stimulation is 37-40 ℃; the temperature of the low-temperature stimulation is 0-15 ℃;
the amino acid comprises one or a combination of at least two of cysteine, methionine or cystine, and the addition amount of the amino acid accounts for 0.1-0.25% of the mass of the culture medium;
(2) Centrifuging the fermentation broth at 3000-6000rpm for 8-12min, filtering with a filter membrane with a pore size of 80-100kDa, concentrating, and filtering with a filter membrane with a pore size of 20-30kDa to obtain extracellular body; mixing the extracellular body with a seaweed polysaccharide solution, and freeze-drying to obtain the yeast-derived extracellular body;
the seaweed polysaccharide solution contains 1-4wt% of seaweed polysaccharide, and the mass ratio of the seaweed polysaccharide solution to the extracellular body is 1 (0.8-1.2).
Other specific point values in the above numerical ranges are selectable, and will not be described in detail here.
In a second aspect, the present invention provides a yeast-derived exosome prepared by the preparation method provided in the first aspect.
In a third aspect, the present invention provides the use of a yeast-derived exosome as provided in the second aspect in a health product, a pharmaceutical product or a cosmetic product.
Compared with the prior art, the invention has the following beneficial effects:
the invention stimulates the yeast cells by adopting the sub-stimulation of the simulation environment, the yeast cells generate stress reaction, secrete high-activity exosomes, and simultaneously amino acid is added, so that the yeast cells can be compounded with other substances to play an antioxidant role more effectively; the extracted exosomes are mixed with saccharides, so that the preservation time can be effectively prolonged without reducing the biological activity; the yeast-derived exosome prepared by the invention has good anti-inflammatory and antioxidant effects, and can promote the regeneration of type I and III collagen.
Drawings
FIG. 1 is an electron micrograph of yeast-derived exosomes prepared in example 1;
FIG. 2 is an electron micrograph of yeast-derived exosomes prepared in comparative example 1.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.
"optional" or "any" means that the subsequently described event or event may or may not occur, and that the description includes both cases where the event occurs and cases where the event does not.
The indefinite articles "a" and "an" preceding an element or component of the invention are not limited to the requirement (i.e. the number of occurrences) of the element or component. Thus, the use of "a" or "an" should be interpreted as including one or at least one, and the singular reference of an element or component includes the plural reference unless the amount clearly dictates otherwise.
The description of the terms "one embodiment," "some embodiments," "exemplarily," "specific examples," or "some examples," etc., herein described means that a specific feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this document, the schematic representations of the above terms are not necessarily for the same embodiment or example.
The reagent sources in the following examples are as follows:
beer yeast: natural medicines and green research institute of Guangdong university of industry;
pichia pastoris: natural medicines and green research institute of Guangdong university of industry;
candida species: garcinia cambogia (GARCINIA CAMBOGIA), supplied by the university of Guangdong Industrial university, natural medicine and Green research institute;
algal polysaccharide: sichuan clover organism technology limited.
Example 1
The present example provides a yeast-derived exosome, which is prepared by the following method:
(1) Inoculating activated Saccharomyces cerevisiae flat plate strain into 25mL triangular conical flask containing 5mL YEPD culture medium, and shake culturing at 28deg.C with 170r/min shaking table for 11 hr to obtain Saccharomyces cerevisiae seed solution;
inoculating activated Pichia pastoris flat bacterial strain into a 25mL triangular conical flask filled with 5mL YEPD culture medium, and shake culturing at 28deg.C with a shaking table at 170r/min for 11h to obtain Pichia pastoris seed liquid;
inoculating activated candida flat-plate strains into a 25mL triangular conical flask filled with 5mL of YEPD culture medium, and shake culturing at 28 ℃ with a shaking table at 170r/min for 11h to obtain candida seed liquid;
(2) Inoculating beer yeast seed solution, pichia pastoris seed solution and candida seed solution into a 1000mL triangular flask filled with 250mL of fresh culture medium according to the ratio of 1:1:1, and performing shake culture at the total inoculum size of 8%, at 28 ℃ and 170r/min, and culturing yeast cells to mid-log phase;
(3) Adding hydrogen peroxide into the culture medium to enable the culture medium to contain 0.2mmol/L hydrogen peroxide, simultaneously adding 0.25% L-cysteine, and continuously culturing for 36h to reach a stationary phase to obtain fermentation liquor;
(4) Centrifuging the fermentation liquor for 10min at a rotation speed of 5000rpm, collecting supernatant, filtering with a filter membrane with a pore diameter of 100kDa, collecting supernatant, concentrating to 1/5 of the original volume, filtering with a filter membrane with a pore diameter of 30kDa, and collecting solids;
(5) Mixing the solid with 3% algal polysaccharide solution with equal mass, and freeze-drying for 24 hours to prepare the yeast-derived exosome.
Example 2
The present example provides a yeast-derived exosome, which is prepared by the following method:
(1) Inoculating activated Saccharomyces cerevisiae flat plate strain into 25mL triangular conical flask containing 5mL YEPD culture medium, and shake culturing at 28deg.C with 170r/min shaking table for 11 hr to obtain Saccharomyces cerevisiae seed solution;
inoculating activated Pichia pastoris flat bacterial strain into a 25mL triangular conical flask filled with 5mL YEPD culture medium, and shake culturing at 28deg.C with a shaking table at 170r/min for 11h to obtain Pichia pastoris seed liquid;
inoculating activated candida flat-plate strains into a 25mL triangular conical flask filled with 5mL of YEPD culture medium, and shake culturing at 28 ℃ with a shaking table at 170r/min for 11h to obtain candida seed liquid;
(2) Inoculating beer yeast seed solution, pichia pastoris seed solution and candida seed solution into a 1000mL triangular flask filled with 250mL of fresh culture medium according to the ratio of 1:0.9:1, wherein the sum of the inoculum sizes is 7%, the temperature is 28 ℃, the speed is 170r/min, and culturing yeast cells to mid-log phase by shaking culture;
(3) Adding potassium chloride into the culture medium to enable the culture medium to contain 0.5mmol/L potassium chloride aqueous solution, simultaneously adding 0.2% cysteine, and continuously culturing for 40 hours until reaching a stable period to obtain fermentation liquor;
(4) Centrifuging the fermentation liquor for 10min at a rotation speed of 5000rpm, collecting supernatant, filtering with a filter membrane with a pore diameter of 90kDa, collecting supernatant, concentrating to 1/4 of the original volume, filtering with a filter membrane with a pore diameter of 20kDa, and collecting solids;
(5) Mixing the solid with 2% algal polysaccharide solution with equal mass, and freeze-drying for 30h to obtain the yeast-derived exosome.
Example 3
The present example provides a yeast-derived exosome, which is prepared by the following method:
(1) Inoculating activated Saccharomyces cerevisiae flat plate strain into 25mL triangular conical flask containing 5mL YEPD culture medium, and shake culturing at 28deg.C with 170r/min shaking table for 11 hr to obtain Saccharomyces cerevisiae seed solution;
inoculating activated Pichia pastoris flat bacterial strain into a 25mL triangular conical flask filled with 5mL YEPD culture medium, and shake culturing at 28deg.C with a shaking table at 170r/min for 11h to obtain Pichia pastoris seed liquid;
inoculating activated candida flat-plate strains into a 25mL triangular conical flask filled with 5mL of YEPD culture medium, and shake culturing at 28 ℃ with a shaking table at 170r/min for 11h to obtain candida seed liquid;
(2) Inoculating beer yeast seed solution, pichia pastoris seed solution and candida seed solution into a 1000mL triangular flask filled with 250mL of fresh culture medium according to the ratio of 1:1:0.8, wherein the sum of the inoculum sizes is 9%, the temperature is 28 ℃, the speed is 170r/min, and culturing yeast cells to mid-log phase by shaking culture;
(3) Raising the temperature in the culture medium to 37 ℃, adding 0.15% L-cysteine, and continuing culturing for 35 hours until reaching a stationary phase to obtain fermentation liquor;
(4) Centrifuging the fermentation liquor for 10min at a rotation speed of 5000rpm, collecting supernatant, filtering with a filter membrane with a pore diameter of 100kDa, collecting supernatant, concentrating to 1/5 of the original volume, filtering with a filter membrane with a pore diameter of 20kDa, and collecting solids;
(5) Mixing the solid with a 4% algal polysaccharide solution with equal mass, and freeze-drying for 30 hours to prepare the yeast-derived exosome.
Example 4
The present example provides a yeast-derived exosome, which is prepared by the following method:
it differs from example 3 only in step (3), and step (3) of this example is:
the temperature in the culture medium is reduced to 10 ℃, 0.15 percent of L-cysteine is added, and the culture is continued for 35 hours until reaching the stationary phase, thus obtaining the fermentation liquor. The other raw materials, amounts and preparation methods were the same as in example 1.
Example 5
The present example provides a yeast-derived exosome, which is prepared by the following method:
it differs from example 1 only in steps (1) and (2), steps (1) and (2) of this example being in order:
(1) Inoculating activated Saccharomyces cerevisiae flat plate strain into 25mL triangular conical flask containing 5mL YEPD culture medium, and shake culturing at 28deg.C with 170r/min shaking table for 11 hr to obtain Saccharomyces cerevisiae seed solution;
inoculating activated Pichia pastoris flat bacterial strain into a 25mL triangular conical flask filled with 5mL YEPD culture medium, and shake culturing at 28deg.C with a shaking table at 170r/min for 11h to obtain Pichia pastoris seed liquid;
(2) The beer yeast seed solution and the Pichia pastoris seed solution are inoculated into a 1000mL triangular flask filled with 250mL of fresh culture medium according to the ratio of 1:1, the sum of the inoculation amount is 8 percent, the temperature is 28 ℃, the speed is 170r/min, and the yeast cells are cultivated to mid-log phase by shaking cultivation. The other raw materials, amounts and preparation methods were the same as in example 1.
Example 6
The present example provides a yeast-derived exosome, which is prepared by the following method:
it differs from example 1 only in steps (1) and (2), steps (1) and (2) of this example being in order:
(1) Inoculating activated Saccharomyces cerevisiae flat plate strain into 25mL triangular conical flask containing 5mL YEPD culture medium, and shake culturing at 28deg.C with 170r/min shaking table for 11 hr to obtain Saccharomyces cerevisiae seed solution;
inoculating activated candida flat-plate strains into a 25mL triangular conical flask filled with 5mL of YEPD culture medium, and shake culturing at 28 ℃ with a shaking table at 170r/min for 11h to obtain candida seed liquid;
(2) Beer yeast seed solution and candida seed solution are inoculated into a 1000mL triangular flask filled with 250mL of fresh culture medium according to the ratio of 1:1, the sum of the inoculation amount is 8 percent, the temperature is 28 ℃, the speed is 170r/min, and the yeast cells are cultivated to mid-log phase by shaking culture. The other raw materials, amounts and preparation methods were the same as in example 1.
Example 7
The present example provides a yeast-derived exosome, which is prepared by the following method:
it differs from example 1 only in steps (1) and (2), steps (1) and (2) of this example being in order:
(1) Inoculating activated Pichia pastoris flat bacterial strain into a 25mL triangular conical flask filled with 5mL YEPD culture medium, and shake culturing at 28deg.C with a shaking table at 170r/min for 11h to obtain Pichia pastoris seed liquid;
inoculating activated candida flat-plate strains into a 25mL triangular conical flask filled with 5mL of YEPD culture medium, and shake culturing at 28 ℃ with a shaking table at 170r/min for 11h to obtain candida seed liquid;
(2) Pichia pastoris seed solution and candida seed solution are inoculated into a 1000mL triangular flask filled with 250mL of fresh culture medium according to the ratio of 1:1, the sum of the inoculation amount is 8 percent, the temperature is 28 ℃, the speed is 170r/min, and the yeast cells are cultivated to mid-log phase by shaking cultivation. The other raw materials, amounts and preparation methods were the same as in example 1.
Example 8
The present example provides a yeast-derived exosome, which is prepared by the following method:
it differs from example 1 only in steps (1) and (2), steps (1) and (2) of this example being in order:
(1) Inoculating activated Saccharomyces cerevisiae flat plate strain into 25mL triangular conical flask containing 5mL YEPD culture medium, and shake culturing at 28deg.C with 170r/min shaking table for 11 hr to obtain Saccharomyces cerevisiae seed solution;
(2) Beer yeast seed solution was inoculated into 1000mL triangular flask containing 250mL fresh medium at an inoculum size of 8%,28℃at 170r/min, and cultured by shaking to mid-log phase. The other raw materials, amounts and preparation methods were the same as in example 1.
Example 9
The present example provides a yeast-derived exosome, which is prepared by the following method:
it differs from example 1 only in steps (1) and (2), steps (1) and (2) of this example being in order:
(1) Inoculating activated Pichia pastoris flat bacterial strain into a 25mL triangular conical flask filled with 5mL YEPD culture medium, and shake culturing at 28deg.C with a shaking table at 170r/min for 11h to obtain Pichia pastoris seed liquid;
(2) Pichia pastoris seed solution is inoculated into a 1000mL triangular flask filled with 250mL of fresh culture medium, the inoculum size is 8 percent, the temperature is 28 ℃, the speed is 170r/min, and the yeast cells are cultivated to mid-logarithmic phase by shaking cultivation. The other raw materials, amounts and preparation methods were the same as in example 1.
Example 10
The present example provides a yeast-derived exosome, which is prepared by the following method:
it differs from example 1 only in steps (1) and (2), steps (1) and (2) of this example being in order:
(1) Inoculating activated candida flat-plate strains into a 25mL triangular conical flask filled with 5mL of YEPD culture medium, and shake culturing at 28 ℃ with a shaking table at 170r/min for 11h to obtain candida seed liquid;
(2) The candida seed solution is inoculated into a 1000mL triangular flask filled with 250mL of fresh culture medium, the inoculation amount is 8 percent, the temperature is 28 ℃, the speed is 170r/min, and the yeast cells are cultivated to mid-logarithmic phase by shaking culture. The other raw materials, amounts and preparation methods were the same as in example 1.
Comparative example 1
This comparative example provides a yeast-derived exosome, which is prepared as follows:
it differs from example 1 only in step (3), and step (3) of this comparative example is:
adding L-cysteine accounting for 0.25% of the mass of the culture medium into the culture medium, and continuing to culture for 36h until reaching a stable period to obtain fermentation liquor. The other raw materials, amounts and preparation methods were the same as in example 1.
Comparative example 2
This comparative example provides a yeast-derived exosome, which is prepared as follows:
it differs from example 1 only in step (3), and step (3) of this comparative example is:
adding hydrogen peroxide into the culture medium to enable the culture medium to contain 0.2mmol/L hydrogen peroxide, and continuously culturing for 36 hours until reaching a stable period to obtain fermentation liquor. The other raw materials, amounts and preparation methods were the same as in example 1.
Test example 1
Repair effect of yeast-derived exosomes on hydrogen peroxide-damaged NIH-3T3 cells
Mouse NIH-3T3 cells were cultured in complete medium containing 10% FBS fetal bovine serum, 1% diabody (streptomycin 100mg/mL, penicillin 100U/mL) and 90% DMEM. After NIH-3T3 cells are cultured to be full, digesting, centrifuging and counting to prepare 5 ten thousand/mL cell suspension; inoculating 100 μl of cell suspension per well in a 96-well plate (at a density of 5000 cells/well); after 24H incubation, the medium was removed, washed 1 time with PBS solution, and 100. Mu.L of 600. Mu.mol/mL H was added 2 O 2 Solution (original concentration 100mM, volume accounting for +2mL depending on sample plate requirement); after 2h, the hydrogen peroxide was aspirated, and the mixture was gently washed 1 time with 120. Mu.L of PBS, and 100. Mu.L of yeast cell exogenesis was added to the administration groupThe body solution, model group, negative control group, blank group added with complete medium containing 10% FBS; after 48h, the extract solution was aspirated, and after 1 wash with 120. Mu.L PBS, 100. Mu.L MTT solution (0.5 mg/mL) was added; (original MTT preparation concentration is 5mg/mL, subpackaging at-20 ℃ in dark place for preservation, and accounting for +2mL according to the required amount of a sample plate); after 4h, the MTT solution was poured out (without washing with PBS), 150. Mu.L of DMSO solution was added, incubated for 10min, OD value was measured by shaking for 20s,490nm or 570nm using an ELISA, and relative viability of the cells was calculated, wherein the yeast cell exosome solutions representing the yeast-derived exosomes prepared in examples 1-10 and comparative examples 1-2 were dissolved in PBS, respectively, to obtain 1.6mg/mL and 0.4mg/mL solutions.
Relative viability% of cells = (OD 1-OD 3)/(OD 2-OD 3) ×100%,
OD1: absorbance values for stress/control group;
OD2: absorbance values for the blank control group;
OD3: absorbance values for the zeroed set.
The relative viability of NIH-3T3 cells obtained from yeast-derived exosomes prepared in examples 1-10, comparative examples 1-2 is shown in Table 1.
Test example 2
Inhibition of RAW264.7 cell NO secretion by yeast-derived exosomes
The mouse macrophage cell line RAW264.7 was cultured in complete medium containing 10% FBS fetal bovine serum, 1% diabody (streptomycin 100mg/mL, penicillin 100U/mL) and 90% DMEM. After RAW264.7 cells are cultured to be full, adding 2mL of complete culture medium, scraping the cells by a cell scraper, uniformly collecting the scraped cells into a centrifuge tube by blowing, centrifuging at 1000rpm for 3min, performing digestion and centrifugation counting, preparing 2 ten thousand/mL of cell suspension, and inoculating 100 mu L of cell suspension (2000 cells/hole) into each hole of a 96-well plate; culturing for 12-20h until the cells adhere to the wall;
the medium was aspirated from each well of the 96-well plate, the blank, the negative control, and 1 column of 1640 complete medium containing 10% FBS was left as the model group, and the administration group was prepared with 10% complete medium according to the type and concentration of the drug, and 100 μl of complete medium containing the drug was added to each group. Placing the 24-pore plate into an incubator, and continuously culturing for 24 hours; cell culture for about 24 hours, medium of each well on the 96-well plate was aspirated, a blank group, a negative control group was added with 1640 complete medium containing 10% FBS, a model group, and a dosing group was added with 1 μg/mL LPS solution; placing the 24-pore plate into an incubator, and continuously culturing for 24 hours; sucking 50 mu L of supernatant into a 96 plate, detecting the NO content according to a NO reagent, and testing the absorbance value (OD) of an absorbance microplate reader at 540nm wavelength; and (3) data processing and calculation, namely calculating the OD values of the cells after the samples to be measured with different concentrations are processed. And (5) making a standard curve, and calculating the NO content of the treatment group. Wherein the administration group represented yeast-derived exosomes prepared in examples 1-10 and comparative examples 1-2.
The NO content X is calculated as follows (Y is the peak area):
Y＝(0.0074)X+(0.0778)，R 2 ＝0.9996。
the NO content obtained by the yeast-derived exosome tests prepared in examples 1 to 10 and comparative examples 1 to 2 is shown in Table 1.
Test example 3
Promotion of NIH-3T3 cell secretion of extracellular matrix collagens I and III by Yeast derived exosomes
(1) After NIH-3T3 cells were cultured to confluence, the cells were counted by digestion centrifugation to prepare 5 ten thousand/mL cell suspension, and 100. Mu.L of cell suspension (5000 cells/well) was seeded per well in 96-well plates; culturing for 12-20h until the cells adhere to the wall;
(2) Separating out culture medium, washing the plate with PBS for 1 time, adding 100 μl of yeast-derived exosome solution prepared in examples 1-10 and comparative examples 1-2 diluted with complete culture medium into the drug group, respectively, adding complete culture medium containing 10% FBS into the model group and blank group, and culturing in incubator for 24 hr or 48 hr;
(3) Sucking the culture solution in the 96-well plate into an EP tube with the volume of 0.5mL, centrifuging at 12000r/min and the temperature of 4 ℃ for 10min (taking care of opening a centrifuge for precooling in advance), and collecting the supernatant;
(4) The level of murine type I and type III collagen in the specimens was determined using a double antibody sandwich method. Coating a microplate with purified mouse type I and type III collagen capture antibodies to prepare a solid-phase antibody, sequentially adding mouse type I and type III collagen into the coated microwells, combining with an HRP-labeled detection antibody to form an antibody-antigen-enzyme-labeled antibody complex, and thoroughly washing and then adding a substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and to final yellow under the action of acid. The shade of the color was positively correlated with murine type I, type III collagen in the sample. And measuring absorbance at a wavelength of 450nm by using an enzyme-labeled instrument, and calculating the contents of the type I collagen and the type III collagen of the mice in the sample by using a standard curve. The test results are shown in Table 1.
Test example 4
Electron microscope experiment
Transmission electron microscopy (transmission electron microscopy, TEM) is a widely used nano-microscopy imaging technique, often used for characterization of EVs, and can identify and analyze exosomes from both morphology and size aspects. Identification principle: the accelerated and focused electron beam is projected onto a very thin sample, and the electrons collide with atoms in the sample to change direction, thereby generating solid angle scattering. The magnitude of the scattering angle is related to the density, thickness, etc. of the sample, so that images with different shades can be formed, which are displayed on an imaging device (such as a fluorescent screen, a film, and a photosensitive coupling member) after being enlarged and focused. In the process of imaging the EVs by using TEM, heavy metal salts such as osmium tetroxide and uranium acetate are often used for carrying out negative staining on the EVs to highlight the lipid membrane structure of the EVs, and the test example carries out staining scanning electron microscope observation of TEM by uranium acetate, and an electron microscope observation condition of the yeast-derived exosomes prepared in example 1 is shown in fig. 1, and an electron microscope observation condition of the yeast-derived exosomes prepared in comparative example 1 is shown in fig. 2; as shown in the figure, the sizes of the exosomes prepared in example 1 and comparative example 1 are about 100nm, and the exosomes have the structural characteristics of a double-layer capsule structure, a shape and a size composite exosomes.
TABLE 1
The yeast-derived exosomes prepared in examples 1-4 can repair NIH-3T3 cells damaged by hydrogen peroxide oxidation, show good antioxidant bioactivity, and the synergistic effect of the three yeasts improves the bioactivity of the exosomes, so that when two yeasts or a single yeast is used, the survival rate of the NIH-3T3 cells damaged by hydrogen peroxide oxidation is greatly reduced; when the simulation environment sub-stimulus is not carried out in the mid-log period of the saccharomycetes, the survival rate of NIH-3T3 cells damaged by hydrogen peroxide oxidation is obviously reduced; when amino acid is not added in the mid-log phase of the saccharomycete, the survival rate of NIH-3T3 cells damaged by hydrogen peroxide oxidation is obviously reduced.
The yeast-derived exosomes prepared in examples 1-4 were able to inhibit the production of NO by LPS-induced macrophages, had good anti-inflammatory bioactivity, and the synergistic effect of the three yeasts increased the bioactivity of the exosomes, with significantly reduced inhibition of NO production by macrophages when two yeasts or a single yeast was used; when the mid-log phase of the saccharomycete is not stimulated by the simulation environment, the inhibition effect on NO generated by macrophages is obviously weakened; when NO amino acid is added in the mid-log phase of yeast, the inhibition of NO production by macrophages is significantly reduced.
The yeast-derived exosomes prepared in examples 1-4 were able to significantly promote the production of extracellular matrix collagen I and collagen III, the synergistic effect of the three yeasts increased the biological activity of the exosomes, and when two yeasts or a single yeast was used, the production of extracellular matrix collagen I and collagen III was significantly reduced; when the simulation environment sub-stimulus is not carried out in the mid-log phase of the saccharomycete, the generation of the extracellular matrix collagen I and the extracellular matrix collagen III is obviously weakened; when no amino acid is added in mid-log phase of yeast, the production of the extracellular matrix collagen I and collagen III is significantly reduced.
Therefore, the yeast-derived exosome provided by the invention has good antioxidant and anti-inflammatory effects, and can be applied to the fields of medicines, health products or cosmetics and the like related to relieving inflammatory reaction and delaying skin aging.
The applicant states that the process of the invention is illustrated by the above examples, but the invention is not limited to, i.e. does not mean that the invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
Claims (14)
1. A method for preparing yeast-derived exosomes, comprising the steps of:
(1) When yeast is cultured to a logarithmic phase, performing simulated environment sub-stimulus treatment, adding amino acid, and then continuously culturing to a stationary phase to obtain fermentation liquor;
(2) Carrying out solid-liquid separation on the fermentation liquor to obtain an extracellular body; mixing the extracellular exosomes with a saccharide to obtain the yeast-derived exosomes;
wherein the yeast is a combination of beer yeast, pichia pastoris and candida, and the inoculation ratio of the beer yeast, the pichia pastoris and the candida is 1 (0.8-1.2) to 0.8-1.2;
the simulation environment sub-stimulus comprises any one of oxidative stress stimulus, high osmotic pressure stimulus, high temperature stimulus or low temperature stimulus, and the reagent adopted by the oxidative stress stimulus is hydrogen peroxide; the high osmotic pressure stimulation adopts a saline solution, wherein the saline solution comprises a potassium chloride aqueous solution; the temperature of the high-temperature stimulation is 37-40 ℃; the temperature of the low-temperature stimulation is 0-15 ℃; the amino acids include cysteine;
the solid-liquid separation comprises the steps of centrifuging the fermentation liquor for 8-12min at the rotating speed of 3000-6000rpm, performing primary filtration by adopting a filter membrane with the aperture of 80-100kDa, concentrating, and performing secondary filtration by adopting a filter membrane with the aperture of 20-30kDa to obtain the extracellular body.
2. The method according to claim 1, wherein the yeast is inoculated in an amount of 6 to 10%.
3. The method of claim 1, wherein the time from the culturing to the log phase is 8-15 h.
4. The method according to claim 1, wherein the time for continuing the culture to the stationary phase is 30-45 h.
5. The method according to claim 1, wherein the temperature at which the cultivation is carried out in the logarithmic phase and the cultivation is continued in the stationary phase in the step (1) is each independently 25 to 30 ℃.
6. The method according to claim 1, wherein H in the medium is at the time of oxidative stress stimulation 2 O 2 The content of (C) is 0.08-0.25 mmol/L.
7. The method according to claim 1, wherein the concentration of the salt in the medium upon high osmotic pressure stimulation is 0.3 to 0.8mmol/L.
8. The preparation method according to claim 1, wherein the amino acid is added in an amount of 0.1 to 0.25% by mass of the culture medium.
9. The method of claim 1, wherein the saccharide is a algal polysaccharide solution.
10. The preparation method according to claim 9, wherein the algal polysaccharide in the algal polysaccharide solution is 1-4% by mass.
11. The method according to claim 9, wherein in the step (2), the mass ratio of the algal polysaccharide solution to the extracellular body is 1 (0.8-1.2).
12. The method according to claim 1, wherein in the step (2), the step of freeze-drying is further included after the mixing.
13. The preparation method according to claim 1, characterized in that the preparation method comprises the steps of:
(1) Culturing yeast at 25-30deg.C for 8-15h, performing simulation environment sub-stimulus treatment until log phase, adding amino acid, and continuously culturing for 30-45h until stable phase to obtain fermentation broth;
the inoculation amount of the yeast is 6-10%, the yeast is a combination of beer yeast, pichia pastoris and candida, and the inoculation ratio of the beer yeast, the pichia pastoris and the candida is 1 (0.8-1.2): 0.8-1.2);
the simulation environment sub-stimulus comprises any one of oxidative stress stimulus, high osmotic pressure stimulus, high temperature stimulus or low temperature stimulus, wherein the reagent adopted by the oxidative stress stimulus is hydrogen peroxide, and H in the culture medium is used during the oxidative stress stimulus 2 O 2 The content of (2) is 0.08-0.25mmol/L; the high osmotic pressure stimulation adopts a saline solution, wherein the saline solution comprises a potassium chloride aqueous solution, and the concentration of salt in a culture medium is 0.3-0.8mmol/L during the high osmotic pressure stimulation; the temperature of the high-temperature stimulation is 37-40 ℃; the temperature of the low-temperature stimulation is 0-15 ℃;
the amino acid comprises cysteine, and the addition amount of the amino acid accounts for 0.1-0.25% of the mass of the culture medium;
(2) Centrifuging the fermentation broth for 8-12min at 3000-6000rpm, filtering with a filter membrane with a pore size of 80-100kDa, concentrating, and filtering with a filter membrane with a pore size of 20-30kDa to obtain extracellular body; mixing the extracellular body with a seaweed polysaccharide solution, and freeze-drying to obtain the yeast-derived extracellular body;
the seaweed polysaccharide solution contains 1-4wt% of seaweed polysaccharide, and the mass ratio of the seaweed polysaccharide solution to the extracellular body is 1 (0.8-1.2).
14. A yeast-derived exosome, characterized in that it is prepared by the preparation method according to any one of claims 1 to 13.
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Inventor after: Wang Juan Inventor after: Peng Yi Inventor after: Xiao Yuan Inventor after: Lin Li Inventor after: Guo Chaowan Inventor after: Hu Lu Inventor before: Wang Juan Inventor before: Peng Yi Inventor before: Xiao Huan Inventor before: Lin Li Inventor before: Guo Chaowan Inventor before: Hu Lu |